Summary/Abstract: The ribosome is one the most ubiquitously conserved and best characterized of the multi- component cellular RNPs. While much is known about ribosome structure and function, there are still many details to be elucidated especially regarding ribosome assembly and its impact the functional capacity of ribosomes. Thus, our work to address biosynthesis of E. coli small ribosomal subunits (SSUs) will have a significant impact on understanding not only assembly and function of these particles but also on the dynamic assembly and function of other RNPs. Moreover approximately 40% of the total energy production of E. coli is consumed to synthesize ribosomal components. This observation and the direct correlation between the rates of E. coli growth and ribosome biogenesis indicate that an accurately assembled and functional ribosome is of utmost importance for cell viability. Our long term goal is to gain a detailed understanding of ribosome biogenesis and thus reveal the impact of this dynamic process on fundamental aspects of cell physiology, such as growth regulation. This proposal details studies of E. coli SSU biogenesis. The purpose of this work is to gain an in-depth understanding of conformational changes and the factors that modulate these changes during the course of functional SSU assembly. Three specific aims have been proposed to address these questions. First, conformational changes within 16S rRNA and specific rRNA nucleotides and ribosomal proteins that facilitate these changes during different stages of assembly in vitro and in vivo will be identified. Second, using a combination of genetics and biochemistry, novel SSU assembly intermediates will be identified and characterized. Third, auxiliary factors that facilitate SSU assembly will be analyzed. Factors identified in the current funding period will be explored in detail and additional factors will be identified and characterized. Our progress during the current funding cycle and our preliminary data indicate that all three specific aims will be successful and thus will directly impact understanding of SSU biogenesis and greatly expand understanding of RNA-protein interactions and RNP assembly in general. By revealing aspects that are critical for both assembly and function of SSUs, we may identify novel target sites for antimicrobial action. Given the growing prevalence of antibiotic resistant bacterial strains, the discovery of such targets is critical for human health.